Summary
Ⅳ Discussion
We introduced a chimeric Wiv-1 construct, TOMSSF 5' ::Wiv-1, into 'Micro-Tom' (Fig. 1). No detrimental effects on plant growth, such as those found in tobacco transformants that expressed a yeast invertase gene driven by the constitutive 35S promoter (Sonnewald et al., 1991), were observed in the transformants. The level of Wiv-1 mRNA was positively correlated with hexose content in young fruits of the transformants (Table 1, Fig. 4).
Why did we observe positive correlations among the hexose contents and the Wiv-1 mRNA levels in young fruits of the transformants? It is known that the sugar unloading process involves two pathways: sucrose from sieve elements enters the sink organs with extracellular hydrolysis (apoplastic pathway via hexose transporters) or without extracellular hydrolysis (mainly the passive symplastic pathway via plasmodesmata) by CWI (Roitsch and Gonzalez, 2004; Ruan and Patrick, 1995). Several authors have suggested that an apoplastic pathway using CWI is involved in the unloading process in tomato fruits (Jin et al., 2009; McCurdy et al., 2010; Ruan and Patrick, 1995; Zanor et al., 2009). The absence of correlation of Wiv-1 and Lin5 mRNAs suggests that no compensatory changes in the level of Lin5 mRNA had occurred in the transformants. It is likely that most of the fluctuation in the level of Wiv-1 mRNA in fruits of the transformants (Fig. 3) was caused by overexpression of the mRNA driven by the TOMSSF 5' promoter (Fig. 2). The promoter of the tomato SS gene TOMSSF is strongly expressed in the vascular tissues of young fruits (Ohyama et al., 2010). Considering the present results together with previously published data, we suggest that the positive correlations between the hexose and the Wiv-1 mRNA levels observed in young fruits of the TOMSSF 5' ::Wiv-1 transformants (Table 1, Fig. 4) were probably achieved by promotion of the apoplastic hexose transport during the early stages of fruit development, through an increase in sucrose degradation activity in the apoplasts of vascular tissues (probably phloem). The correlations between the mRNA level and the hexose contents in young fruits of transformants were higher at lower levels of Wiv-1 mRNA (Table 1, Fig. 4), which suggests that extreme expression of Wiv-1 is not necessary to increase fruit hexose content. The fact that CWI is an enzyme with a low Michaelis constant, Km (Karuppiah et al., 1989), may support this hypothesis. Furthermore, the lack of correlations between the levels of Wiv-1 mRNA
Fig. 4 Scatterplot of the relative expression level of Wiv-1 (based on mRNA levels;
see also Table 1) and hexose content in young fruits transformed with the TOMSSF 5' ::Wiv-1 construct.
r: correlation coefficient; *Significant at P < 0.05.
大山ら ： 細胞壁結合型酸性インベルターゼ遺伝子Wiv-1の果実特異的発現によるトマト果実糖蓄積への影響（英文） 41
and the sucrose content (Table 1) may indicate that most of the sucrose transported into the fruit's cytosol is derived from the symplastic pathway without extracellular hydrolysis.
Even though the apoplastic pathway is active in young fruit, it's unclear why there was no correlation among the hexose contents and the natural fluctuation of Lin5 mRNA levels in young fruits of the transformants. One possibility is that the TOMSSF promoter (used for the construct) drives more favorable gene expression than does the native promoter of Lin5.
One limitation of our study is that the results were obtained only from the T0 generation of transformants. To further characterize the sugar accumulation mechanisms in tomato fruit, it will be necessary to re-evaluate the transformed lines for both their mRNA levels and their enzyme levels after the transgenes are fixed (made homozygous). It will also be necessary to introduce the TOMSSF 5' ::Wiv-1 construct into normal tomato cultivars, rather than dwarf cultivars such as 'Micro-Tom'. Furthermore, in addition to the young-fruit-specific promoter, it will be necessary to analyze the effects of a ripening-related phloem promoter construct to analyze the effects of CWI on sugar content in ripening fruits.
Summary
Because soluble sugars are an important component of tomato fruit quality, we investigated the possible role of a key enzyme, cell-wall-bound acid invertase (CWI), in sugar accumulation in tomato fruit. We fused cDNA for Wiv-1, a gene encoding tomato CWI, to a young-fruit-specific tomato sucrose synthase (SS) gene promoter, and introduced the chimeric construct into tomato ('Micro-Tom'). Significant positive correlations were found between the level of Wiv-1 expression and the hexose content in young developing fruits of the transformants. These results suggest that the hexose transport in young fruits was promoted by the expression of Wiv-1, probably through an increase in the level of sucrose degradation in the apoplast.
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細胞壁結合型酸性インベルターゼ遺伝子 Wiv-1 の 果実特異的発現によるトマト果実糖蓄積への影響
大山 暁男 ・ 布目 司 ・ 福岡 浩之
摘 要
糖含量はトマト果実の品質を左右する重要な要素である． そこで我々は， トマト果実の糖蓄積における鍵酵素の 1つ， 細胞壁結合型酸性インベルターゼ （CWI） の生理的役割について調査した． 細胞壁結合型酸性インベル
ターゼcDNA Wiv-1を， 若い果実に特異的な発現を示すトマトショ糖合成酵素遺伝子プロモーターと連結し， 得
られたキメラ遺伝子をトマト （マイクロトム） に導入した． その結果， 形質転換体の若い果実においては，
Wiv-1発現レベルと還元糖量との間に有意な正の相関が見出された． 以上の結果は， 形質転換体の若い果実におい
て，Wiv-1遺伝子の発現に起因するアポプラスト部のショ糖分解レベルの増大を介して果実中への還元糖輸送が促進
されたことを示唆している．
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〒514-2392 三重県津市安濃町草生360 野菜育種・ゲノム研究領域
平成25年9月27日受理
43 43 野菜茶業研究所研究報告 13：43～47（2014）
日本産及び外国産の紅茶の含水率
池田 奈実子
（平成25年9月30日受理）